关键词: 草地贪夜蛾, 劳氏粘虫, 信息素结合蛋白, 原核表达, 荧光竞争结合

Abstract: 【Aim】 This study aims to determine the binding characteristics of three pheromone binding proteins (PBPs) from the fall armyworm, Spodoptera frugiperda with sex pheromone and gland components of S. frugiperda and sympatric Leucania loreyi, and thus to investigate the roles of the three SfruPBPs in the recognition of pheromone components of these two species. 【Methods】 Plasmids were constructed using pET-30a(+) as the prokaryotic expression vector of the three SfruPBPs of S. frugiperda and introduced into Escherichia coli. After the OD6₀₀ value of the culture of E. coli reached 0.6-0.8, the three SfruPBPs were induced to express using IPTG, and purified by the Ni-NTA magnetic agarose beads. The binding characteristics of the three SfruPBPs with 14 sex pheromone and gland components of S. frugiperda and L. loreyi were assayed by fluorescence competitive binding assay. 【Results】 The recombinant PBP proteins SfruPBP1-3 were obtained by prokaryotic expression. Single target protein bands of expected size were obtained in the agarose gel electrophoresis after protein purification. Fluorescence competitive binding assay revealed that among the reported 14 sex pheromone and gland components of S. frugiperda and L. loreyi, SfruPBP1 had strong specific binding ability with the major pheromone component Z9-14∶Ac (Ki=0.80 μmol/L) of these two pest species, and SfruPBP2 displayed broad ligand spectrum, showing strong binding ability with the minor pheromone component Z7-12∶Ac and the gland components 12∶Ac, E7-12∶Ac and Z10-14∶Ac (Ki<1.00 μmol/L), and medium binding ability with the major pheromone component Z9-14∶Ac and the gland components Z9-12∶Ac and 11-12∶Ac (1.00 μmol/L<Ki<4.00 μmol/L). SfruPBP3 had medium binding ability only with the gland component Z9-12∶Ac, with the Ki value of 1.36 μmol/L. Among the chemicals with strong or medium binding affinities to SfruPBP, Z9-14∶Ac, Z7-12∶Ac and 12∶Ac were also sex pheromone or gland components of L. loreyi, however, all the three SfruPBPs showed no obvious binding ability with Z7-14∶Ac, a specific gland component in L. loreyi. 【Conclusion】 All the three SfruPBPs of S. frugiperda play important roles in the sex pheromone perception, but each SfruPBP displays a distinct selectivity to components. The three SfruPBPs show no obvious binding ability with the specific gland component Z7-14∶Ac of L. loreyi.

Key words: Spodoptera frugiperda, Leucania loreyi, pheromone binding protein, prokaryotic expression, fluorescence competitive binding